Name: optimizing sample preparation process for transmission electron microscopy of neuromuscular junctions in drosophila larvae
Uploaded: 2023-09-15T14:00:00
Description: Watch this Scientific Journal Video about Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae at JoVE.com

这项工作得到了中国自然科学基金32070811和东南大学（中国）分析测试基金11240090971的支持。我们感谢中国南京东南大学医学院电子显微镜实验室和形态分析中心。

注：本文中使用的透射电子显微镜样品制备方法之前已报道过16.需要注意的是，试剂的选择和剂量的调整是必要的，具体取决于样品。在样品制备过程中使用有毒化学试剂很多，因此，操作人员需要采取一定的防护措施，如穿防护服和手套，在通风橱中操作。
1. 解剖、固定和放置程序
<ol><li>解剖<ol><li>在Jan溶液中解剖游荡的3龄后期幼虫，并根?...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BMP+homolog+Gbb+provides+a+retrograde+signal+that+regulates+synaptic+growth+at+the+Drosophila+neuromuscular+junction.">The BMP homolog Gbb provides a retrograde signal that regulates synaptic growth at the Drosophila neuromuscular junction.</a> Neuron. 39 (2), 241-254 (2003).</li><li>Guangming, G., Mei, C., Chenchen, Z., Wei, X., Junhua, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+analysis+method+of+neuromuscular+junction+in+Drosophila.+larvae+by+transmission+electron+microscopy.">Improved analysis method of neuromuscular junction in Drosophila. larvae by transmission electron microscopy.</a> Anatomical Science International. 97 (1), 147-154 (2022).</li><li>Brent, J. R., Werner, K. M., McCabe, B. 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果蝇 幼虫样品在脱水过程中容易卷曲，因为样品很薄，难以准确定位神经肌肉接头，从而增加了样品制备的难度和工作量。传统的改进是缩短了样品7个，但样品仍进行了不同程度的卷曲。
在我们的方法中，有两个关键步骤：首先，由于金属网的限制，样品在整个脱水过程中保持平坦。此外，在聚合过程中，可以通过将样品夹在塑料制品之间来聚合样...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64934">Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae</a>. The Authors section was updated from:
Gan Guangming1,2 
Sheng Qingyuan3 
Ou Yutong1 
Chen Mei1 
Zhang Chenchen1 
1The School of Medicine, Southeast University, 
2The Key Laboratory of Developmental Genes and Human Disease, Southeast University, 
3The School of Life Science and Technology, Southeast University 
Corresponding Authors:&#160;Gan Guangming
to:
Gan Guangming1,2 
Sheng Qingyuan3 
Ou Yutong1 
Chen Mei3 
Zhang Chenchen1 
1The School of Medicine, Southeast University, 
2The Key Laboratory of Developmental Genes and Human Disease, Southeast University, 
3The School of Life Science and Technology, Southeast University 
Corresponding Authors:&#160;Gan Guangming,&#160;Sheng Qingyuan

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64934">Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae</a>. The Authors section was updated.

Erratum: Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

电子显微镜是研究生物材料超微结构最理想的方法之一，可以在纳米级1级直观准确地展示细胞的内部结构。然而，由于样品制备过程的复杂性和高昂的成本，电子显微镜不如光学显微镜那么受欢迎。电子显微镜技术的最新进展导致了图像质量的显着提高，同时显着减少了相关的工作量。因此，电子显微镜在推进不同领域的科学知识方面发挥着重要作用2.
果蝇是一种优秀的动物模型，用于执行遗传操作以精确控制靶基因的空间和时间表达3。此外，与哺乳动物模型相比，果蝇具有生长期短、易于饲养的优点;因此，果蝇在形态学研究中被广泛使用4,5。
在果蝇幼虫中，神经肌肉接头 （NMJ） 胸膜广泛分布在肌肉中 6,7，NMJ 的免疫染色可以很容易地提供有关突触数量和形态的信息 8,9 。位于 A2 和 A 3 节段的第 6/7节肌肉中的 NMJ 胸肌非常适合使用光学显微镜进行定量和形态学研究。这是因为它们的大小和丰度10,11。因此，果蝇幼虫 NMJ 被认为是神经科学研究的有用模型12。
然而，通过透射电镜观察NMJ bouton超微结构具有挑战性。由于透射电子显微镜的扫描窗口较窄，因此很难定位广泛分布的NMJ boutons13。另一个原因是，在样品制备方案7的酒精脱水步骤中，果蝇体壁容易卷曲。
传统研究通常选择 A2 和 A 3 节段的第 6 和第7 块肌肉之间的胸针作为样品材料，因为它们的丰度和大小14,15。A2 和 A 3 节段的第 6 和第7 节肌肉比其他肌肉大，并且包含更多的胸花。然而，当制备用于电子显微镜的样品时，固定样品往往会变薄并且容易卷曲，从而导致 A2 和 A3 节的第 6 和第7 块肌肉定位不当。
我们在此报告一种新的处理程序，与传统的样品制备方法相比，该程序更有效地防止样品卷曲7,16，通过允许样品在随后的脱水过程中保持平坦，从而促进果蝇幼虫神经肌肉接头的更好定位。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-Epoxypropane</td>
			<td>SHANGHAI LING FENG CHEMICAL REAGENT CO., LTD</td>
			<td>JYJ 037-2015</td>
			<td>Penetrating Agent</td>
		</tr>
		<tr>
			<td>Drosophila Stocks</td>
			<td>Bloomington</td>
			<td>none</td>
			<td>The wild-type control Drosophila strains used in this research were all W1118, and were reared according to standard culture methods</td>
		</tr>
		<tr>
			<td>Flat-bottomed glass test tubes</td>
			<td>Haimen Chenxing Experimental equipment Company&#160;</td>
			<td>none</td>
			<td>Flat-bottomed glass test tubes(bottle)with sponge plug(or bottle stopper)</td>
		</tr>
		<tr>
			<td>K4M cross-linker</td>
			<td>&#160;Agar Scientific</td>
			<td>Cat# 1924B</td>
			<td>The embedding resins are based on a highly cross-linked acrylate and methacrylate formula&#160;</td>
		</tr>
		<tr>
			<td>K4M resin (monomer B)</td>
			<td>&#160;Agar Scientific</td>
			<td>Lot# 631557</td>
			<td>Resin Monomer</td>
		</tr>
		<tr>
			<td>Polyvinyl film</td>
			<td>Haimen Chenxing Experimental equipment Company&#160;</td>
			<td>none</td>
			<td>Transparent polyethylene film is the best , thickness of about 0.2mm</td>
		</tr>
		<tr>
			<td>SPI Chem DDSA</td>
			<td>SPI</td>
			<td>SpI#02827-AF</td>
			<td>Dodecenyl Succinic Anhydride</td>
		</tr>
		<tr>
			<td>SPI-Chem DMP-30 Epoxy</td>
			<td>SPI</td>
			<td>02823-DA</td>
			<td>Accelerator</td>
		</tr>
		<tr>
			<td>SPI-Chem NMA</td>
			<td>SPI</td>
			<td>SpI#02828-AF</td>
			<td>Hardner for Epoxy</td>
		</tr>
		<tr>
			<td>SPI-PON 812 Epoxy</td>
			<td>SPI</td>
			<td>SPI#0259-AB</td>
			<td>Resin Monomer</td>
		</tr>
		<tr>
			<td>Steel mesh&#160;</td>
			<td>Yuhuiyuan Gardening Store(online)</td>
			<td>none</td>
			<td>Copper or stainless steel net</td>
		</tr>
		<tr>
			<td>Transmission electron microscopy</td>
			<td>Hitachi H-7650</td>
			<td>11416692</td>
			<td>All grids (on which samples were gathered) were stained with lead citrate and observed under a transmission electron microscopy.</td>
		</tr>
		<tr>
			<td>Ultrathin microtome</td>
			<td>Leica UC7 ultrathin microtome</td>
			<td>595915</td>
			<td>All sectioning operations are carried out on a Leica UC7 ultrathin microtome using a diamond knife</td>
		</tr>
	</tbody>
</table>

optimizing sample preparation process for transmission electron microscopy of neuromuscular junctions in drosophila larvae

果蝇神经肌肉接头 （NMJ） 已成为神经科学领域有价值的模型系统。在果蝇 NMJ 中应用共聚焦显微镜使研究人员能够获取突触信息，包括突触丰度的定量数据和对其形态的详细见解。然而，透射电镜的漫射分布和有限的视觉范围给超微结构分析带来了挑战。本研究引入了一种创新且高效的样品制备方法，该方法超越了传统方法。该过程首先将金属网放在平底瓶或试管的底部，然后将固定的幼虫样品定位到网状物上。在样品上放置一个额外的网格，确保它们位于两个网格之间。在进行包埋程序之前，将固定样品彻底脱水并渗透。然后以平片方式将样品嵌入环氧树脂中，这允许准备用于定位和切片的肌肉。受益于这些步骤，果蝇幼虫的所有肌肉都可以在光学显微镜下看到，从而促进后续的定位和切片。在定位身体节段 A2 和 A3 的第 6 和第 7块肌肉后，去除多余的树脂。进行第 6或第 7块肌肉的连续超薄切片。

果蝇幼虫体壁由30根可识别的肌肉纤维组成，这些肌肉纤维以规则的模式排列，在解剖和固定后看起来像一片薄片21（图1A）。由于金属网的存在，样品在脱水过程中保持平坦（图1B，C）。将幼虫体肌肉埋在由环氧树脂制成的薄板中（图1D-G），该薄板易于准确地定位并...

Watch this Scientific Journal Video about Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae at JoVE.com

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

本报告提供了一种新的样品制备程序，用于可视化 果蝇 幼虫中的神经肌肉接头。与传统方法相比，该方法在防止样品卷曲方面更有效，对于 果蝇 神经肌肉接头超微结构分析特别有用。

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

The Drosophila neuromuscular junction (NMJ) has emerged as a valuable model system in the field of neuroscience. The application of confocal microscopy at ...

透射电镜的漫射分布和有限的视觉范围给基础设施分析带来了挑战。该协议提供了一种创新且高效的样品制备方法，超越了传统方法。与传统方法相比，这种先进的TEM样品制备方法在防止样品卷曲方面更有效，因为它允许样品在随后的脱水过程中保持平坦。根据样品类型，必须有必要选择试剂和调整剂量。样品制备过程中会使用许多有毒化学试剂。演示该程序的是来自甘光明博士实验室的硕士生盛清源。首先，在Jan溶液中解剖游荡的三龄果蝇幼虫，并按照标准程序获得整个体壁。用固定剂在解剖室中以四摄氏度固定幼虫体壁过夜。第二天，将幼虫体壁从解剖室转移到1.5ml离心管中，并用二甲胂酸钠缓冲液冲洗数次。将幼虫体壁用1%四氧化锇固定两小时。固定后，用蒸馏水洗涤样品数次。将 2% 饱和尿液醋酸溶液加入试管中，以染色 PHI 神经肌肉接头。两小时后，用去离子水冲洗样品。接下来用剪刀剪下两片孔径为 270 微米的圆形金属网。将金属网放在平底瓶的底部。然后将固定的幼虫放在网上，然后放置另一个网眼。在4摄氏度的温度下，将样品在增加的乙醇浓度下脱水20分钟。然后用环氧丙烷在室温下冲洗样品两次，每次五分钟。接下来，将样品置于环氧丙烷和环氧树脂溶液中，然后在室温下将纯环氧树脂置于两小时。为了包埋，将聚乙烯薄膜切成一个大正方形和一个小的空心矩形支架。将小方块与大方块粘合在一起。在大方块的中心加入足够的环氧树脂。然后打开立体镜，将样品放入预先放置的环氧树脂中，肌肉侧朝上。在样品上加入几滴环氧树脂，并用聚乙烯薄膜覆盖它们。将用于聚合的样品在升高的温度下放置 24 小时。聚合后，使用锋利的刀片去除幼虫体的多余部分，同时保留梯形，包括 A2 和 A3 段。从剩余的幼虫体壁上取下梯形，并用AB胶将其连接到填充树脂的胶囊上。在光学显微镜下，确认 A2 和 A3 节段的第六块和第七块肌肉的位置，保持切线平面平行于第六块肌肉。然后沿 A3 段的两侧切去五分之一的样品。使用切片机，从第六块肌肉开始制备样品的超薄切片。切片后，将尽可能多的切片连接到每个铜网上。使用透射电子显微镜观察样品。在Lowicryl K4M树脂和环氧树脂中，Lowicryl K4M树脂提供了更清晰，更容易观察到果蝇身体肌肉的结果。共聚焦成像增强了第六块和第七块肌肉之间的神经肌肉接头的可视化。透射电子显微镜显示珠状神经肌肉接头，提供改进的突触结构信息。由于金属网的限制，样品在整个脱水过程中保持平坦。此外，样品在塑料产品之间聚合，防止样品折叠。未来，TEM样品制备方法可以应用于脑和腹侧神经索神经桩，从而提高超微结构研究的潜力。

Research

JoVE Journal

Neuroscience

Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

聚焦离子束铣削和脑组织的扫描电子显微镜

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

科研

神经科学

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

Appetitive Associative Olfactory Learning in Drosophila Larvae

欲求联想嗅觉学习<em&gt;果蝇</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

Foraging Path-length Protocol for Drosophila melanogaster Larvae

觅食路径长度协议<em&gt;果蝇</em&gt;幼虫

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Novel Assay for Cold Nociception in Drosophila Larvae

Novel Assay for Cold Nociception in Drosophila Larvae

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

提耳伸肌压钳条件下哺乳动物神经肌肉传输的检查准备

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...

Preparation of the Rat Vocal Fold for Neuromuscular Analyses

用于神经肌肉分析的大鼠声带的制备

The purpose of this tutorial is to describe the preparation of the rat vocal fold for histochemical neuromuscular study. This protocol outlines procedures ...

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

在微流体装置中生成具有功能性神经肌肉接头的人类运动单元

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

优化 果蝇 幼虫神经肌肉接头透射电子显微镜样品制备工艺

优化果蝇幼虫神经肌肉接头透射电子显微镜样品制备工艺